Amyloid-beta peptide binds to microtubule-associated protein 1B (MAP1B)

Abstract

Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer's disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer's disease.

Fig. 1

PAGE and western blot analysis of recombinant phage expressing the fragment of MAP1B. 10¹¹ phage particles diluted in loading buffer were resolved on 4–12% NuPAGE Bis-Tris gel (Invitrogen) at 200 V for 45 min at room temperature and immunoblotted for detection with anti-pIII and anti-E-tag antibodies. Wild-type M13 phage was used as a control. Migration of the molecular mass standards as well as pIII and MAP1B-pIII fusion protein are indicated by arrowheads. Note, that pIII band in the wild-type M13 phage has a slightly high MW compared with the pIII band in recombinant phagemid C8 due to deletions present in the cloning vector.

Fig. 2

Analysis of interaction of Aβ1-42 with C8 phage bearing the fragment of MAP1B. Phage concentration used was 10¹¹ per ml, and 100 μl were added to each well. M13 phage and a non-related peptide (NRP) were used as negative controls. OD at 405 was registered. Data are means of three independent experiments.

Fig. 3

Double immunofluorescence staining of Aβ and MAP1B in differentiated SY5Y cells. Differentiated cells were fixed and doubly immunostained with monoclonal mouse anti-human Aβ17-24 antibody (B and F) and polyclonal goat anti-human MAP1B-HC (A) or rabbit anti-human MAP1B-LC antibodies (E). The primary antibodies were visualized with AlexaFluor 594 (green) anti-rabbit, AlexaFluor 594 (green) anti-goat or AlexaFluor 488 (red) anti-mouse antibodies. When anti-MAP1B-HC antibody was used, in merged image (C) and merged image at higher magnification (D) colocalizations of Aβ and MAP1B appear yellow. When staining of the cells was performed with anti-human MAP1B-LC antibody, in merged images (G) and merged image at higher magnification (H) no colocalizations were observed. Scale bar - 40 μm.